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Biomedical subjects

J Tam

Publications and source records attributed to J Tam.

At least 19 recordsLinked to original sources
PubMed2003

Disrupted circadian rhythms in VIP- and PHI-deficient mice.

The related neuropeptides vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are expressed at high levels in the neurons of the suprachiasmatic nucleus (SCN), but their function in the regulation of circadian rhythms is unknown. To study the role of these peptides on the circadian system in vivo, a new mouse model was developed in which both VIP and PHI genes were disrupted by homologous recombination. In a light-dark cycle, these mice exhibited diurnal rhythms in activity which were largely indistinguishable from wild-type controls. In constant darkness, the VIP/PHI-deficient mice exhibited pronounced abnormalities in their circadian system. The activity patterns started approximately 8 h earlier than predicted by the previous light cycle. In addition, lack of VIP/PHI led to a shortened free-running period and a loss of the coherence and precision of the circadian locomotor activity rhythm. In about one-quarter of VIP/PHI mice examined, the wheel-running rhythm became arrhythmic after several weeks in constant darkness. Another striking example of these deficits is seen in the split-activity patterns expressed by the mutant mice when they were exposed to a skeleton photoperiod. In addition, the VIP/PHI-deficient mice exhibited deficits in the response of their circadian system to light. Electrophysiological analysis indicates that VIP enhances inhibitory synaptic transmission within the SCN of wild-type and VIP/PHI-deficient mice. Together, the observations suggest that VIP/PHI peptides are critically involved in both the generation of circadian oscillations as well as the normal synchronization of these rhythms to light.

Animals↗
PubMed2002

Chronic estrogen deficiency leads to molecular aberrations related to neurodegenerative changes in follitropin receptor knockout female mice.

The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen deficiency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen's known pleiotropic effects in the nervous system, our study examined the effects of genetically induced estrogen-testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-beta-estradiol (E2) in FORKO mice are significantly decreased (FORKO -/-: 1.13+/-0.34 pg/ml; wild-type +/+: 17.6+/-3.5 pg/ml, P<0.0001, n=32-41); in contrast, testosterone levels are increased (-/-: 37.7+/-2.3 pg/ml; wild-type +/+: 3.9+/-1.7 pg/ml, P<0.005, n=25-33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neurofilament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50+/-3%, hippocampus 24+/-2%, cortex 12+/-3%) and in DRGs (11+/-6%). Moreover, we observed aberrations in the enzymatic activities of mitogen-activated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20-50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the findings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system.

Acetylcholine↗
PubMed2002

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat.

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Animals↗
PubMed2002

Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis.

Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are central mediators of the apoptotic program. Here we demonstrate that caspases cleave and inactivate the plasma membrane Ca(2+) pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca(2+) handling, which results in Ca(2+) overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca(2+) handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.

Animals↗
PubMed2001

Embryonic expression of pituitary adenylyl cyclase-activating polypeptide and its selective type I receptor gene in the frog Xenopus laevis neural tube.

The genes encoding pituitary adenylyl cyclase-activating peptide (PACAP) and its selective type I receptor (PAC1) are expressed in the embryonic mouse neural tube, where they may be involved in neurogenesis and neural tube development. We examined here the early expression and potential actions of PACAP and PAC1 in the vertebrate developmental model Xenopus laevis. PACAP and PAC1 mRNAs were first detected by RT-PCR in stage 16-18 embryos (18 hours after fertilization). Two distinct PACAP precursor mRNAs were identified. One encoded both growth hormone-releasing hormone and PACAP, whereas the other encoded only full-length PACAP. Unlike that in the adult, the latter represented the predominant embryonic PACAP mRNA species. In situ hybridization revealed that PACAP and PAC1 mRNAs were restricted to neural cells. PAC1 gene expression was observed mainly in the ventricular zone in the ventral parts of the prosencephalon, mensencephalon, rhombencephalon, and anterior spinal cord. In contrast, PACAP mRNA was localized exclusively in postmitotic cells in the dorsolateral parts of the rhombencephalon and entire spinal cord. Most PACAP mRNA-containing cells were characterized as Rohon-Beard neurons. Exposure of early embryos to UV irradiation, which ventralizes embryos and inhibits neural induction, reduced the expression of PACAP and PAC1 genes. These results suggest that PACAP may be involved in the early development of the embryonic Xenopus neural tube.

Animals↗
PubMed2001

Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis.

Caspase activation and proteolytic cleavage of specific target proteins represents an integral step in the pathway leading to the apoptotic death of cells. Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of apoptosis. In an effort to develop a strategy with which to monitor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase substrates that display a quantifiable change in their spectral properties when cleaved by group II caspases. Specifically, tandem GFP substrates linked by a caspase-sensitive cleavage site show diminished fluorescence resonance energy transfer (FRET), as a consequence of cleavage, due to physical separation of the GFP moieties in apoptotic cells. We have evaluated the influence of different caspase-sensitive linkers on both FRET efficiency and cleavage by caspase-3. We also demonstrate that caspase activity as well as inhibition by pharmacological agents can be monitored, with minimal manipulation, in intact adherent cells seeded in a 96-well cell culture dish. Finally, we have adapted this technology to a high throughput screening platform to identify novel small molecule and cell permeable inhibitors of apoptosis. Based on a biochemical analysis of the compounds identified it is clear that this assay can be used to detect drugs which inhibit caspases directly as well as those which target upstream components of the caspase cascade.

Apoptosis↗
PubMed2000

Molecular cloning of growth hormone-releasing hormone/pituitary adenylyl cyclase-activating polypeptide in the frog Xenopus laevis: brain distribution and regulation after castration.

Pituitary adenylyl cyclase-activating peptide (PACAP) appears to regulate several neuroendocrine functions in the frog, but its messenger RNA (mRNA) structure and brain distribution are unknown. To understand the potential role of PACAP in the male frog hypothalamic-pituitary-gonadal axis, we cloned the frog Xenopus laevis PACAP mRNA and determined its distribution in the brain. We then analyzed the castration-induced alterations of mRNA expression for PACAP and its selective type I receptor (PAC1) in the hypothalamic anterior preoptic area, a region known to regulate reproductive function. The PACAP mRNA encodes a peptide precursor predicted to give rise to both GH-releasing hormone and PACAP. The deduced peptide sequence of PACAP-38 was nearly identical to that of human PACAP with one amino acid substitution. Abundant PACAP mRNA was detected in the brain, but not several other tissues, including the testis. In situ hybridization revealed strong expression of the PACAP gene in the dorsal pallium, ventral hypothalamus, and nuclei of cerebellum. PACAP mRNA signals were weak to moderate in the hypothalamic anterior preoptic area and were absent in the pituitary. Castration induced an increase in the expression of PACAP and PAC1 receptor mRNAs in the hypothalamic anterior preoptic area after 3 days. Replacement with testosterone prevented the castration-induced changes. These results provide a molecular basis for studying the physiological functions of PACAP in frog brain and suggest that PACAP may be involved in the feedback regulation of hypothalamic-pituitary-gonadal axis.

Amino Acid Sequence↗
PubMed2000

Baseline blink rates and the effect of visual task difficulty and position of gaze.

PURPOSE: To compare the baseline blink rates (BBR) measured under different (baseline) conditions, and to compare the blink rates measured when performing two visual tasks of different levels of difficulty at two positions of gaze. METHODS: In the first single masked experiment, BBR were measured under three different conditions - (i) conversation with the subjects (ii) taking the visual acuity and (iii) keeping the subjects waiting in an empty examination room. In the second single masked experiment, the subjects were required to perform an easy task (reading normal English words) and a difficult task (reading mirror-image English words) at primary gaze and down gaze. The orders of the tasks performed were randomly allocated. RESULTS: BBR taken under the three different conditions were significantly different from each other. No significant difference was found between the blink rates measured when performing the two reading tasks if they were performed at the same position of gaze. The mean blink rates were significantly lower when performing the tasks at down gaze than when performing the tasks at primary gaze. No significant differences were found between BBR-waiting and blink rate measured when the subject was doing the easy task at primary gaze, and between BBR-VA and blink rate measured when the subject was performing the difficult task at primary gaze. CONCLUSIONS: BBR measured under different (baseline) conditions produced different results so it is important for investigators to describe the baseline condition very clearly in studies where BBR are measured. Our results also indicated that mean blink rate was affected by the position of gaze and not the level of task difficulty.

Adult↗
PubMed1999

Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus.

It has been demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) promotes the survival of neurons in culture and can inhibit neuronal cell death after experimental injury. Furthermore, peripheral axotomy results in increased PACAP gene expression in sensory and sympathetic neurons, suggesting that PACAP might be a mediator in the injury response in certain parts of the nervous system. However, changes in PACAP expression have not been reported in injured motor neurons, despite the significant problem of motor neuron degeneration in injury and in several neurological diseases. We examined here changes in gene expression of PACAP and two high-affinity PACAP receptors, PAC(1) and VPAC(2), in adult rat motor neurons after facial nerve axotomy by in situ hybridization. PACAP gene expression was very low in facial motor neurons of normal rats. However, a robust time-dependent increase in PACAP mRNA was observed in the facial motor nucleus in most or all axotomized motor neurons. This induction was detectable 6 hr after axotomy, and peaked at 48 hr, when expression on the injured side averaged more than 20-fold higher than that on the contralateral side. Thereafter, PACAP mRNA levels decreased slightly, but remained more than 10-fold elevated for as long as 30 days after axotomy. In contrast to PACAP, gene expression for both the PAC(1) and VPAC(2) receptor was high in facial motor neurons of normal rats. No significant change was observed for VPAC(2) receptor gene expression in facial motor neurons after axotomy, whereas gene expression for the PAC(1) receptor became significantly decreased. The results indicate that the PACAP ligand receptor system is tightly regulated in the facial motor nucleus after axotomy, providing evidence that PACAP may be involved in motor injury responses.

Animals↗
PubMed1999

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Animals↗
PubMed1999

Delay effect in a model for virus replication.

As biology becomes more quantitative, it appears that the increasing use of mathematics in this area is inevitable. In 1996, Nowak & Bangham (1996, Science 272, 74-79) proposed three mathematical models to explore the relation between antiviral immune responses, virus load, and virus diversity. In this paper we investigate the delay effect in a model which considers the interaction between a replicating virus and host cells. We assume that there is a finite time lag between infection of a cell and the emission of viral particles. Even with the introduction of this delay, the steady states of the model--as suggested by Nowak & Bangham--remain stable. The result also gives a condition for how the parameter values should be chosen when analysing clinical data so that the model remains tenable.

Humans↗
PubMed1996

Use of generic antibiotics in children.

Most oral antibiotics have highly acceptable generic equivalents. The generic products are comparable in bioavailability, side-effect profile, and efficacy to their brand-name equivalents. Some are more palatable, and all are less costly that the trade-name products. Increased use of oral generic antibiotics will generate significant cost savings for both the patients and prescribers, without compromising therapeutic benefits.

Anti-Bacterial Agents↗
PubMed1995

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Animals↗
PubMed1995

Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule.

Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.

Amino Acid Sequence↗
PubMed1994

The influence of adenine-rich motifs in the 3' portion of the ribosome binding site on human IFN-gamma gene expression in Escherichia coli.

The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon. Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well. Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs. Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene. The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated. We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation. We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths. The results suggest a positive functional role for the 3' adenine-rich motif. A possible mechanism for these effects is discussed.

Adenine↗
PubMed1993

Systemic absorption of salbutamol following nebulizer delivery in acute asthma.

We measured plasma salbutamol concentrations in 35 children with acute asthma attacks before and after nebulizer therapy. The main finding was that older children had higher concentrations than younger children, despite similar dosage regimens. There was no influence of severity of the asthma attack on the following measures: pre- and post-nebulizer concentrations, absolute and percentage change in salbutamol levels with therapy. We conclude that the theoretical risks of high concentrations in young children were not found and we suggest that the very young may indeed require higher doses due to possible problems in nebulizer technique or differences in kinetics.

Absorption↗
PubMed1992

In vivo chemical footprinting of the Escherichia coli ribosome.

We have studied the in vivo chemical accessibility of 16S rRNA residues A349-G1505 in the small subunit of the Escherichia coli ribosome. Exponentially growing E. coli cultures were reacted with dimethyl sulfate, and the reactive sites on the 16S ribosomal RNA were analyzed by reverse transcription, an assay which detects reactions at N1-A and N3-C. In agreement with previous in vitro results, three regions of 16S RNA appeared particularly reactive to dimethyl sulfate: hairpin 27 (residues A892-A915) of the central domain, and hairpin 33-33A (residues A994-C1037) and the tip of hairpin 41 (residues A1256-A1275), both from the 3' major domain. These three regions contained 52% of the reactive residues but only 8% of the residues scanned. In contrast to previous in vitro results, three small sections of 16S RNA appeared protected: the tip of hairpins 26 (residue A845) and 31 (residues A968-A969), and residues A1418, A1441, and A1483 of the middle body of hairpin 44. Four of the dimethyl sulfate reactive sites (A831, C948, A1019, and C1192) are located in positions usually assumed to be double-stranded (helices 26, 30, 33-33A, and 34), which suggests alternative structures for these helices at least during part of the translation process, as if the residues in question belonged to "conformational switches." The addition of chloramphenicol protected residues A831, A1035-A1036, and A1503, which suggests that they belong to the mobile regions of the elongating ribosome, and become exposed during some transition(s) from one ribosomal state to the other during the elongation cycle.

Adenosine↗